Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Determination of optimal temperature(s) in juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) based on growth performance and stress responses

This study sought to determine the optimal temperature(s) for aquaculture of juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) (mean initial BW: 3.1 g). Growth performance, insulin‐like growth factor 1 (IGF‐1) expression and thermal stress responses (plasma cortisol, glucose, and hepatic heat shock protein 60 expression) were evaluated at three constant temperatures (24°C, 26°C and 28°C) in a 2‐week trial. At the end of the trial, final BW was significantly higher at 26°C and 28°C than at 24°C (p < 0.05); a quadratic regression analysis of final BW showed the optimum temperature for growth was 27.5°C (p < 0.05, R² = 0.806). The highest hepatic IGF‐1 expression was observed at 26°C (p < 0.05). On the other hand, hepatic heat shock protein 60 expression was highest at 28°C (p < 0.05), suggesting thermal stress. In conclusion, temperature optima, which support excellent growth but induce minimal thermal stress, was 26°C. This fine information within a narrow temperature range is expected to give empirical information for red‐spotted grouper farmers to sustain maximal production efficiency with avoiding thermal stress and to determine the future location of production, especially in consideration of arising seawater temperatures.     

Beneficial co‐culture of jellyfish Rhopilema esculenta (Kishinouye) and sea cucumber Apostichopus japonicus (Selenka): implications for pelagic‐benthic coupling

This study investigated monthly changes of sedimentation and sediment properties in three different culture systems (ponds) – i.e. jellyfish Rhopilema esculenta monoculture (J), sea cucumber Apostichopus japonicus and jellyfish co‐culture (SJ) and sea cucumber monoculture (S) – to verify the feasibility of co‐culturing jellyfish and sea cucumbers. Results showed that jellyfish culture accelerated the settling velocity of total particulate matter (TPM). Average TPM settling velocities in the SJ (75.6 g m^?2 day^?1) and J (71.1 g m^?2 day^?1) ponds were significantly higher than that in the S pond (21.7 g m^?2 day^?1) from June to September during the jellyfish culture period. Average settling velocities of organic matter (OM), total organic carbon (TOC), total nitrogen (TN) and total phosphorus (TP) in the SJ pond increased significantly by 3.0, 2.9, 3.3 and 3.8 times, respectively, compared with those in the S pond. Sediment contents of OM, TOC, TN and TP in the SJ and J ponds were significantly higher than those in the S pond during the jellyfish culture season. The specific growth rate of sea cucumbers feeding on SJ sediment was significantly higher than that of those feeding on S sediment. Co‐culturing sea cucumbers with jellyfish may help alleviate benthic nutrient loading due to the jellyfish and provide a secondary cash crop.     

Preliminary assessment of large‐scale co‐culture of sandfish (Holothuria scabra) with the Babylon snail (Babylonia areolata) in earthen ponds and in raceways

Holothuria scabra (sandfish) and Babylonia areolata were trialed in two large‐scale co‐culture experiments. Experiment 1 assessed co‐culture in 4 × 400 m² earthen ponds where Babylonia were cultured in a central pen at a density of 400 individuals/m² with sandfish occupying the remaining pond space at 1.1 individuals/m². Sandfish grew from 18.20 ± 6.67 g to 119.03 ± 17.74 g in 92 days and Babylonia (fed trash fish in both experiments) grew from 0.90 ± 0.38 g to 4.93 ± 1.44 g, but Babylonia growth was not increased in co‐culture compared to monoculture. Water and sediment quality varied between co‐culture and monoculture ponds. Neither showed clear improvement due to sandfish culture. Experiment 2 compared non‐segregated sandfish‐Babylonia co‐culture with Babylonia monoculture in 20 m² concrete raceways. Sandfish were cultured at 2 individuals/m² and Babylonia at 300 individuals/m². Sandfish grew up to 1.91 g.day^?1 with 100% survival. Babylonia weight gain was significantly greater in co‐culture raceways (3.35 ± 0.64 g over 61 days), which was double that of Babylonia in monoculture. Substrate total N was reduced by 20% in co‐culture compared with monoculture (p = .032). This provisional study of commercial scale sandfish‐Babylonia co‐culture demonstrates culture compatibility, providing a basis for further system development.     

Selecting and ranking areas for conservation of Aegla (Crustacea: Decapoda: Anomura) in southern South America integrating biogeography,phylogeny and assessments of extinction risk

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Effects of dietary protein and lipid levels on growth,feed utilization and body composition in red‐tailed catfish juveniles (Hemibagrus wyckioides,Chaux & Fang 1949)

A feeding trial was conducted in a recycling water system during 10 weeks to determine the optimal protein to lipid ratio in Asian red‐tailed catfish (Hemibagrus wyckioides). Six diets of two protein levels (390 and 440 g kg^?1) with three lipid levels (60, 90 and 120 g kg^?1) were formulated. Fish (1.96 g) were fed six diets with four replicates to apparent satiation at a stocking density of 50 fish per tank (500 L). Faeces were collected in cultured tanks at the end of the feeding trial for digestibility measurement. Significantly, improved growth performances (P < 0.01) and higher feed utilization (P < 0.001) were observed in fish fed with higher lipid diets. However, higher protein diets did not significantly improve fish growth but they reduced FCR (P < 0.001) and protein efficiency ratio (P < 0.01). Higher lipid diets also resulted in significantly increased adipose‐somatic index, carcass fat and reduced moisture of the fish. The study revealed the protein sparing effect of dietary lipid in the catfish and highest growth performance was found by fish fed 390 g kg^?1 protein and 120 g kg^?1 lipid diet with P/E ratio of 20.48 mg protein kJ^?1. DP/DE ratio for maximal growth rate in diets was 21.48 mg protein kJ^?1.     

A review of the Leon springs pupfish (Cyprinodon bovinus) long‐term conservation strategy and response to habitat restoration

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Re‐evaluation of marine benthic species of nature conservation importance: a new perspective on certain ‘lagoonal specialists’ with particular emphasis on Alkmaria romijni Horst (Polychaeta: Ampharetidae)

1. The ampharetid polychaete Alkmaria romijni (protected under the Wildlife and Countryside Act 1981) is recorded from a survey of intertidal mudflats on the south bank of the Humber Estuary (UK) in an apparently unusual habitat for the species. 2. The records from the Humber are considered together with other records which have been collated recently as part of a review of rare and scarce marine benthic species in the UK. Some of these records are ‘new’. 3. An assessment of records from the Humber and elsewhere suggests that this tidal estuarine habitat is not atypical for A. romijni. Furthermore, it is not clear why the species shows such a restricted distribution within the Humber. 4. A. romijni can now be shown to not be confined to brackish lagoons in the UK, but rather is found in muddy sediments in sheltered to very sheltered conditions in both lagoons and certain estuaries. The majority of current records are now estuarine in the UK, suggesting that A. romijni can no longer be classed as a ‘lagoonal specialist’ species as previously considered. 5. Based on all UK records, the species should be considered ‘scarce’ rather than ‘rare’ using current assessment criteria. On balance, however, it is concluded that it is probably still appropriate to maintain the protected status of A. romijni. 6. Two other ‘lagoonal specialist’ species—the starlet sea anemone Nematostella vectensis and the lagoon sand shrimp Gammarus insensibilis—indicate, in the light of new records, that the reappraisal of lagoonal specialist status is restricted, for the present, to A. romijni rather than being common to other species. 7. The work highlights that there is a need for a re‐evaluation mechanism in the assessment of marine species considered to be of conservation importance. Copyright © 2000 John Wiley & Sons, Ltd.     

Validation of environmental DNA (eDNA) as a detection tool for at‐risk freshwater pearly mussel species (Bivalvia: Unionidae)

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Integrating local ecological knowledge,citizen science and long‐term historical data for endangered species conservation: Additional records of angel sharks (Chondrichthyes: Squatinidae) in the Mediterranean Sea

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Cyprinid herpesvirus 3 as a potential biological control agent for carp (Cyprinus carpio) in Australia: susceptibility of non‐target species

Carp (Cyprinus carpio L.) is a pest species in Australian waterways, and cyprinid herpesvirus 3 (CyHV‐3) is being considered as a potential biological control (biocontrol) agent. An important consideration for any such agent is its target specificity. In this study, the susceptibility to CyHV‐3 of a range of non‐target species (NTS) was tested. The NTS were as follows: 13 native Australian, and one introduced, fish species; a lamprey species; a crustacean; two native amphibian species (tadpole and mature stages); two native reptilian species; chickens; and laboratory mice. Animals were exposed to 100–1000 times the approximate minimum amount of CyHV‐3 required to cause disease in carp by intraperitoneal and/or bath challenge, and then examined clinically each day over the course of 28 days post‐challenge. There were no clinical signs, mortalities or histological evidence consistent with a viral infection in a wide taxonomic range of NTS. Furthermore, there was no molecular evidence of infection with CyHV‐3, and, in particular, all RT‐PCRs for viral mRNA were negative. As a consequence, the results encourage further investigation of CyHV‐3 as a potential biocontrol agent that is specific for carp.     

Co‐infection of rainbow trout (Oncorhynchus mykiss) with infectious hematopoietic necrosis virus and Flavobacterium psychrophilum

Co‐infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co‐infection has not been well characterized or documented. In this study, it was hypothesized that fish co‐infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co‐infected with low doses of either IHNV or F. psychrophilum, and at 2 days post‐initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%–100%), while mortality from a single pathogen infection with the same respective dose was low (5%–20%). The onset of mortality was earlier in the co‐infected group (3–4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co‐infection led to a significant increase in the number of F. psychrophilum colony‐forming units and IHNV plaque‐forming units within tissues. This finding confirms that when present together in co‐infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co‐infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co‐infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host–pathogen interactions related to co‐infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.     

Time‐series analysis of native and non‐native crayfish dynamics in the Thames River Basin (south‐eastern England)

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Combined effect of weaning time and co‐feeding duration on growth and survival of peled Coregonus peled (Gmelin) larvae

The study investigated the combined effect of weaning from live feed to a commercial dry pellet at 10, 15, 20, 25 or 30 days posthatching (dph) and co‐feeding for 1, 3 or 6 days on survival and growth of Coregonus peled larvae. Additional groups fed only live Artemia sp. nauplii (ART), and only Biomar LARVIVA ProWean 100 (DRY) were included. A final survival rate of 66.4%–85.5% was observed in groups weaned after 20 dph. Final body weight (BW) and total length (TL) were significantly lower in groups weaned at 10 and 15 dph, regardless of the duration of co‐feeding. Larvae reached 29–37 mg BW and TL of 17.7–19.0 mm in groups weaned at 20, 25 and 30 dph. The recommended minimum duration of feeding with live food, based on these results, is 20 days. Based on the significantly higher yield of larvae weaned after 20 dph irrespective of co‐feeding duration, it can be concluded that abrupt weaning to dry food after 20 days of feeding with live prey can provide adequate production while reducing the effort and costs associated with live feed.     

Use of tissue and sediment‐based threshold concentrations of polychlorinated biphenyls (PCBs) to protect juvenile salmonids listed under the US Endangered Species Act

• 1. Under the Endangered Species Act, the National Marine Fisheries Service has authority to protect listed species from any adverse actions that may jeopardize the population's ability to recover and increase to sustainable levels. Listed salmon species in the northwest United States are known to travel through urban areas in their migration from river to ocean. Species such as the chinook salmon (Oncorhynchus tshawytscha) often spend several weeks in these urban estuaries where they can be highly exposed to urban‐related contaminants that reside in the sediments and accumulate in their prey species. The concern is that these contaminants are bioaccumulated to levels that may impact the ability of individual salmon to grow and mature normally. This paper provides a framework for determining the tissue and sediment concentrations of polychlorinated biphenyls (PCBs) that are likely protective against adverse effects in listed salmonid species.
• 2. The relevant ecotoxicological literature was examined and 15 studies were selected that met the pre‐established criteria outlined here. For each study, the lowest tissue concentration (residue) of total PCBs associated with a biological response was selected. The tissue concentration associated with the 10th percentile of these 15 studies was chosen to represent the residue effect threshold (RET) above which wild juvenile salmonids would be expected to exhibit adverse sublethal effects from accumulated PCBs. This value (2.4 μg PCBs g^?1 lipid) is expressed in terms of the lipid‐normalized concentration because of the large effect lipid can have on the expressed toxicity and the substantial variability in lipid content observed in salmonids over their life cycle.
• 3. A sediment concentration that is expected to produce the RET was then determined using the biota‐sediment accumulation factor approach. The sediment effect threshold, which varies with the total organic carbon content in sediment, is the level above which adverse effects may be expected in juvenile salmonids due to accumulation of PCBs from environmental exposure. Bioaccumulation of PCBs was examined in one river system as a model for determining an appropriate bioaccumulation factor for wild juvenile chinook salmon.
• 4. Evaluation of exposure to potentially deleterious concentrations of PCBs based on tissue residues is the preferred approach; however, the sediment effect threshold may also be used in cases where bioaccumulation has been characterized in an estuary. The threshold values presented here are intended as interim guidelines that should be modified as more data become available. Additionally, because of the uncertainty around many of the factors and assumptions that comprise the single threshold effect values, it is recommended that future studies be employed to help determine a range of acceptable values that would afford protection under various environmental and biological conditions.

Published in 2002 by John Wiley & Sons, Ltd.     

The worth of giants: The consumptive and non‐consumptive use value of the giant sea bass (Stereolepis gigas)

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Revisiting ecological integrity 30 years later: non‐native species and the misdiagnosis of freshwater ecosystem health

Assessing the ecological integrity of freshwater ecosystems has become a priority to protect the threatened biodiversity they hold and secure future accessibility to the services they provide. Some of the most widespread applications of biological indicators are fish‐based indices. These have mostly mirrored the approach proposed by Karr 30 years ago (Index of Biotic Integrity; IBI), based on the comparison of observed and expected composition and structure of local fish assemblages in the absence of major perturbations, using the so‐called reference condition approach. Despite the notable success of the implementation of fish‐based indices, most of them overlook non‐native species as a source of ecosystem degradation, and evaluations are focused on the physico‐chemical condition of freshwater ecosystems and their effects on freshwater biodiversity. Almost 90% of 83 reviewed IBIs did not consider non‐native species when defining reference conditions. Most IBIs used non‐native species in conjunction with native ones to construct the metrics that conform to the index. The response of the IBI to the effect of non‐native species has hardly ever been tested. When developing and evaluating IBIs, attention was mostly directed to ensuring the correct response of the index to physico‐chemical parameters, which could otherwise be characterized more effectively using alternative methods. Current application of IBIs entails a misuse of biological indicators by overlooking some types of degradation that cannot be otherwise evaluated by traditional methods. This constrains the capacity to adequately respond to one of the most challenging and common threats to the conservation of freshwater fish diversity.     

Determining spatial use of the world's second largest humpback dolphin population: Implications for place‐based conservation and management

1. Place‐based management can be an effective conservation tool to protect cetaceans from anthropogenic pressures. The spatial use of the world's second largest population of the threatened Indo‐Pacific humpback dolphins (Sousa chinensis) is not well‐documented, which makes it challenging to designate protected areas for this population.
2. To address this knowledge gap and to test the efficacy of an existing dolphin protected area (Zhanjiang Leizhou Bay Municipal Humpback Dolphin Nature Reserve, ZLBMHDNR), boat‐based surveys were conducted to document dolphin occurrence from 2015 to 2018, covering an area of 1221 km² in the eastern waters off Zhanjiang, China. In total, 253 dolphin group sightings were obtained during 174 survey days.
3. Spatial analysis showed that humpback dolphins aggregated in three core‐use areas with higher sighting density within the survey area. Furthermore, intermediate‐use and broad‐use areas were identified that could be essential for the movements of humpback dolphins among these core‐use areas.
4. The spatial usage of humpback dolphins was compared inside and outside the ZLBMHDNR. Results suggest that the ZLBMHDNR is insufficient to encompass a significant portion of dolphin habitat. The ZLBMHDNR (21 km²) is not large enough, and thus it should be expanded for effective place‐based conservation management in this region.
5. For developing a protected area network, important dolphin habitats identified in this study should be protected as a single management unit. Additionally, mitigation of anthropogenic pressures is needed to be taken into consideration as conservation initiatives in the study area.
6. This study provides support for a more science‐informed protected area network, and highlights the necessity of implementing place‐based conservation and management for the world's second largest humpback dolphin population.

     

Fate and legacy of an invasion: extinct and extant populations of the soft‐shell clam (Mya arenaria) in Grays Harbor (Washington)

1. The soft‐shell clam, Mya arenaria, was intentionally introduced to Washington State during the mid‐1870s. Presently, extensive shell deposits cover parts of the intertidal flats of Grays Harbor, an estuarine ecosystem that has received much attention because of its role as nursery ground for Dungeness crab (Cancer magister), a commercially significant species. 2. Preliminary observations revealed that shells in the deposits belonged to clams that were much larger than extant conspecifics. Shell deposits and extant populations have spatially disjoint distributions; few individuals are found living within the deposits. The purpose of our study was to determine the cause of the different size characteristics between extinct and extant clams, when and how the deposits originated, and why the extant population does not prosper in what appears to be prime habitat. 3. We (i) surveyed extant populations and shell deposits, (ii) followed the extant population through periodic sampling over a 3‐year period, (iii) investigated growth and survival in extant and extinct populations, (iv) experimentally manipulated growth conditions, and (v) searched archival sources for information about the origin of the deposits. 4. We conclude that extinct clams grew faster and lived longer, apparently because they occupied the best habitats available. Archival records suggest that the population expanded rapidly during the period around 20 years after its introduction, but was decimated between 1895 and 1897 by a mass‐mortality episode that resulted in the formation of the deposits. The population since then has never rebounded in its prime habitat, in spite of potential settlers being regularly available. 5. Dungeness crab larvae settle preferentially in the shell deposits, where the abundance of 0+ age juveniles is orders of magnitude higher than in the adjacent flats. We hypothesize that predation by juvenile crabs limits clam recruitment. 6. The introduction of Mya to Grays Harbor during the 1870s has had long‐lasting effects, which persist a century after their once large populations were decimated by a mass‐mortality event. Copyright © 2000 John Wiley & Sons, Ltd.